Project Summary This project explores the development of bicontinuous microemulsions (B?Es) as topical drug delivery systems for antimicrobial peptides (AMPs), as an alternative therapy to treat wound infections. This study addresses the missions of both the NIH?s NIBIB and NIAID research programs through: 1) the development of a novel drug delivery technology, and 2) a new approach to combat chronic wound infections exacerbated by antibiotic-resistant microorganisms. Wound infections are a major problem due to the increased occurrence of antibiotic-resistant microorganisms, which are attributable to $20 billion annually in excess health care costs, $35 billion in societal costs, and 8 million days of extended hospitalization stays in the US. AMPs can kill microbial pathogens that cause wound infections (e.g., methicillin-resistant Staphylococcus aureus [MRSA]) through disruption of negatively charged biomembranes, producing pores that allow leakage of cytoplasmic fluids; however, AMPs must be delivered in a highly folded form to be effective and previous studies have not addressed this need. Thus, this study proposes to develop B?Es as systems for encapsulation of AMPs in their folded state and delivery to wound surfaces. B?Es are optically clear, homogeneous, and thermodynamically stable biomembrane mimetic systems. They possess unique drug-delivery properties compared to other membrane-based systems, including large-volume fractions of water and oil (~40%) that allow co-solubilization of other drugs. Preliminary studies demonstrate that the AMP melittin when encapsulated into B?Es can reside in a highly folded state (>90% ?-helix) and high concentrations (1-10 g/L) are achievable. Several important hypotheses will be tested, including that AMP-loaded B?E solutions are effective antimicrobial agents with activity strongly controlled by the extent of AMP folding. The Specific Aims are to 1) demonstrate that four diverse AMPs can be incorporated into several different biocompatible B?E systems at high (biologically relevant) concentrations and degrees of folding; 2) show that B?Es loaded with AMPs and antiseptic agents such as chlorhexidine (derived in Aim 1) can serve as robust topical preparations for treatment of wound infections. For Aim 1, the relationship between AMP folding and B?E properties will be determined through novel methods, including circular dichroism and small-angle neutron scattering. Aim 2 will provide measurements of minimum inhibitory and bactericidal concentration against several representative microorganisms encountered in wounds (including MRSA), cell cytotoxicity (hemolysis) activity and protection of B?E-encapsulated AMPs from proteolysis. The results will provide a basis for future clinical applications to use B?Es as a drug delivery system for improved activity and/or stability of cell-penetrating peptides. Other applications include adsorption to surfaces of medical devices for antimicrobial coatings and delivery of radiolabeled AMPs for bioimaging.